WO2016089692A1 - Animaux non humains possédant un gène cd47 humanisé - Google Patents

Animaux non humains possédant un gène cd47 humanisé Download PDF

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Publication number
WO2016089692A1
WO2016089692A1 PCT/US2015/062614 US2015062614W WO2016089692A1 WO 2016089692 A1 WO2016089692 A1 WO 2016089692A1 US 2015062614 W US2015062614 W US 2015062614W WO 2016089692 A1 WO2016089692 A1 WO 2016089692A1
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WIPO (PCT)
Prior art keywords
human
rodent
gene
polypeptide
endogenous
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PCT/US2015/062614
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English (en)
Inventor
Cagan Gurer
Ella Ioffe
Alexander MUJICA
Gavin Thurston
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Regeneron Pharmaceuticals, Inc.
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Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=54979926&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=WO2016089692(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Priority to EP20215046.2A priority Critical patent/EP3850946B1/fr
Priority to JP2017526681A priority patent/JP6730277B2/ja
Priority to EP18207075.5A priority patent/EP3466255B1/fr
Priority to DK15813960.0T priority patent/DK3086637T3/en
Priority to RU2017123357A priority patent/RU2728412C2/ru
Priority to KR1020217032415A priority patent/KR102508175B1/ko
Priority to SG11201703463WA priority patent/SG11201703463WA/en
Priority to AU2015355328A priority patent/AU2015355328B2/en
Priority to EP23192469.7A priority patent/EP4296278A3/fr
Priority to CN201580066047.0A priority patent/CN107205368B/zh
Priority to SI201530659T priority patent/SI3086637T1/sl
Priority to EP15813960.0A priority patent/EP3086637B1/fr
Priority to MX2017007293A priority patent/MX2017007293A/es
Priority to BR112017010490A priority patent/BR112017010490A2/pt
Priority to IL286403A priority patent/IL286403B2/en
Priority to PL18207075T priority patent/PL3466255T3/pl
Priority to RS20190370A priority patent/RS58536B1/sr
Priority to KR1020177013948A priority patent/KR102313073B1/ko
Priority to LTEP15813960.0T priority patent/LT3086637T/lt
Priority to PL15813960T priority patent/PL3086637T3/pl
Priority to NZ731471A priority patent/NZ731471A/en
Priority to KR1020237007814A priority patent/KR102617691B1/ko
Priority to CA2967834A priority patent/CA2967834C/fr
Priority to ES15813960T priority patent/ES2716735T3/es
Application filed by Regeneron Pharmaceuticals, Inc. filed Critical Regeneron Pharmaceuticals, Inc.
Publication of WO2016089692A1 publication Critical patent/WO2016089692A1/fr
Priority to IL252019A priority patent/IL252019B/en
Priority to CY20191100334T priority patent/CY1121547T1/el
Priority to HRP20190634TT priority patent/HRP20190634T1/hr
Priority to CY20211100382T priority patent/CY1124089T1/el
Priority to AU2022203787A priority patent/AU2022203787A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Humanized animals, e.g. knockin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • G—PHYSICS
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/5055—Cells of the immune system involving macrophages
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/12—Animals modified by administration of exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A01K2217/052—Animals comprising random inserted nucleic acids (transgenic) inducing gain of function
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0381—Animal model for diseases of the hematopoietic system
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

  • Cancer therapy can be separated into four main categories: chemo/radio therapy, hormone therapy, targeted therapy, and immunotherapy.
  • An intense focus of medical research and development has focused on targeted therapy and significant improvements have been made, yet cancer remains a major challenge to patients and to the healthcare industry worldwide. This major challenge is due, in part, to the ability of cancer cells to evade the monitoring mechanisms of theinnate and adaptive immune systems, which is partly the result of inhibition of phagocytic clearance.
  • Such a system provides a source for assays in phagocytosis and macrophage functions in vivo, and identification of new cancer therapies that are targeted at providing an anti-tumor enviroment by promoting pro-phagocytic signals to the immune system.
  • the present invention encompasses the recognition that it is desirable to engineer non-human animals to permit improved systems for identifying and developing new cancer therapeutics.
  • the present invention also encompasses the recognition that it is desirable to engineer non-human animals to permit improved engraftment of human hematopoietic stem cells.
  • the present invention also encompasses the recognition that non- human animals having a humanized CD47 gene and/or otherwise expressing, containing, or producing a human or humanized CD47 polypeptide are desirable, for example for use in identifying and developing cancer therapeutics that overcome systemic toxicity associated with blockade of CD47 and overcome CD47-mediated inhibition of
  • phagocytosis of tumor cells provide a more efficient in vivo system for engraftment of human hemotopoietic stem cells that provides an increase in homeostasis of a broader number of human cell types.
  • the present invention provides a non-human animal having a genome comprising a CD47 gene that includes genetic material from two different species (e.g., a human and a non-human).
  • the CD47 gene of the non- human animals as described herein encodes a CD47 polypeptide that contains human and non-human portions, wherein the human and non-human portions are linked together and form a functional CD47 polypeptide.
  • a non-human animal of the present invention comprises a CD47 gene that comprises an endogenous portion and a human portion, wherein the endogenous and human portions are operably linked to an endogenous promoter.
  • an endogenous portion comprises exon 1 and the exons downstream of exon 7 of an endogenous CD47 gene.
  • exon 1 and the exons downstream of exon 7 of an endogenous CD47 gene are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the corresponding exon 1 and the exons downstream of exon 7 of a mouse CD47 gene that appears in Table 3.
  • exon 1 and the exons downstream of exon 7 of an endogenous CD47 gene are identical to the corresponding exon 1 and the exons downstream of exon 7 of a mouse CD47 gene that appears in Table 3.
  • a human portion encodes amino acids 16-292 of a human CD47 polypeptide. In some embodiments, a human portion encodes amino acids 19-292 of a human CD47 polypeptide. In some embodiments, a human portion encodes amino acides 19-141 of a human CD47 polypeptide. In some embodiments, a human portion encodes amino acids 19-127 of a human CD47 polypeptide. In some embodiments, a human portion comprises exons 2-7 of a human CD47 gene.
  • exons 2-7 of a human CD47 gene are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the corresponding exons 2-7 of a human CD47 gene that appears in Table 3.
  • exons 2-7 of a human CD47 gene are identical to the corresponding exons 2-7 of a human CD47 gene that appears in Table 3.
  • a non-human animal of the present invention expresses a CD47 polypeptide comprising an extracellular portion of a human CD47 polypeptide and an intracellular portion of an endogenous CD47 polypeptide.
  • a CD47 polypeptide comprises a transmembrane portion of a human CD47 polypeptide.
  • a CD47 polypeptide comprises a transmembrane portion of a non- human CD47 polypeptide.
  • a CD47 polyeptide is translated in a cell of a non-human animal with a non-human signal peptide.
  • a non-human signal peptide is a rodent (e.g., a mouse or a rat) signal peptide.
  • a CD47 polypeptide of the present invention is expressed from an endogenous non-human CD47 gene.
  • an intracellular portion of an endogenous CD47 polypeptide comprises an intracytoplasmic tail that has an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to an intracytoplasmic tail of a mouse CD47 polypeptide that appears in Table 3.
  • an intracellular portion of the endogenous CD47 polypeptide comprises an intracytoplasmic tail that has an amino acid sequence that is identical to an intracytoplasmic tail of a mouse CD47 polypeptide that appears in Table 3.
  • an extracellular portion of a human CD47 polypeptide comprises amino acids corresponding to residues 19-141 of a human CD47 polypeptide. In some embodiments, an extracellular portion of a human CD47 polypeptide comprises amino acids corresponding to residues 19-127 of a human CD47 polypeptide. In some embodiments, the extracellular portion of a human CD47 polypeptide comprises an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to a corresponding amino acid sequence of an extracellular portion of a human CD47 polypeptide that appears in Table 3. In some embodiments, an extracellular portion of a human CD47 polypeptide comprises an amino acid sequence that is identical to a corresponding amino acid sequence of an extracellular portion of a human CD47 polypeptide that appears in Table 3.
  • the present invention provides a CD47 polypeptide encoded by the CD47 gene of a non-human animal as described herein. In some certain embodiments,
  • an encoded CD47 polypeptide comprises an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20.
  • an encoded CD47 polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20.
  • the present invention provides a humanized CD47 gene comprising one or more exons of a non-human CD47 gene operably linked to one or more exons of a human CD47 gene.
  • a humanized CD47 gene of the present invention comprises non-human exons that encode an intracellular portion of a CD47 polypeptide and human exons that encode an extracellular portion of a human CD47 polypeptide.
  • a humanized CD47 gene also comprises human exons that encode a transmembrane portion of a human CD47 polypeptide.
  • a humanized CD47 gene of the present invention comprises non-human exons that encode a signal peptide, in whole or in part, and an intracellular portion of a CD47 polypeptide, and human exons that encode an extracellular portion and optionally a transmembrane portion of a CD47 polypeptide.
  • the present invention provides an isolated cell or tissue from a non-human animal as described herein.
  • the present invention provides an isolated cell or tissue comprising a CD47 gene as described herein.
  • a cell is selected from a dendritic cell, lymphocyte (e.g., a B or T cell), macrophage and a monocyte.
  • a tissue is selected from adipose, bladder, brain, breast, bone marrow, eye, heart, intestine, kidney, liver, lung, lymph node, muscle, pancreas, plasma, serum, skin, spleen, stomach, thymus, testis, ovum, and a combination thereof.
  • the present invention provides a non-human embyronic stem cell whose genome comprises a CD47 gene as described herein.
  • a non-human embyronic stem cell is a mouse embyronic stem cell and is from a 129 strain, C57BL/6 strain or a BALB/c strain.
  • a non-human embyronic stem cell is a mouse embyronic stem cell and is from a mixture of 129 and C57BL/6 strains.
  • the present invention provides the use of a non-human embryonic stem cell as described herein to make a non-human animal.
  • a non-human embryonic stem cell is a mouse embyronic stem cell and is used to make a mouse comprising a CD47 gene as described herein.
  • a non-human embryonic stem cell is a rat embyronic stem cell and is used to make a rat comprising a CD47 gene as described herein.
  • the present invention provides a non-human embyro comprising, made from, obtained from, or generated from a non-human embyronic stem cell comprising a CD47 gene as described herein.
  • a non- human embryo is a rodent embyro.
  • a rodent embryo is a mouse embryo.
  • a rodent embryo is a rat embryo.
  • the present invention provides a method of making a non- human animal that expresses a CD47 polypeptide from an endogenous CD47 gene, wherein the CD47 polypeptide comprises a human sequence, the method comprising inserting a genomic fragment into an endogenous CD47 gene in a non-human embryonic stem cell, said genomic fragment comprising a nucleotide sequence that encodes a human CD47 polypeptide in whole or in part; obtaining a non-human embryonic stem cell comprising an endogenous CD47 gene that comprises the nucleotide sequence that encodes a human CD47 polypeptide in whole or in part; and creating a non-human animal using the non-human embryonic stem cell comprising said nucleotide sequence that encodes a human CD47 polypeptide in whole or in part.
  • a human sequence comprises amino acids corresponding to residues 19-141 (or 19-292) of a human CD47 polypeptide. In some embodiments, a human sequence comprises amino acids corresponding to residues 19-127 of a human CD47 polypeptide.
  • a nucleotide sequence comprises exons 2-7 of a human CD47 gene. In some embodiments, a nucleotide sequence comprises one or more selection markers. In some embodiments, a nucleotide sequence comprises one or more site-specific recombination sites.
  • the method further comprises a step of inserting a genomic fragment into an endogenous SIRPa gene of a non-human embryonic stem cell, said genomic fragment comprising a nucleotide sequence that encodes a human SIRPa polypeptide in whole or in part (e.g., encodes an extracellular portion of a human SIRPa polypeptide).
  • a genomic fragment comprising a nucleotide sequence that encodes a human SIRPa polypeptidein whole or in part e.g., encodes an extracellular portion of a human SIRPa polypeptide
  • the method further comprises breeding a non-human animal comprising an endogenous CD47 gene that includes a nucleotide sequence that encodes a human CD47 polypeptide, in whole or in part, with a second non-human animal, said second non-human animal having a genome comprising a SIRPa gene that encodes a SIRPa polypeptide comprising an extracellular portion of a human SIRPa polypeptide (e.g., amino acids corresponding to residues 28-362 of a human SIRPa polypeptide) and an intracellular portion of an endogenous SIRPa polypeptide.
  • a non-human animal comprising an endogenous CD47 gene that includes a nucleotide sequence that encodes a human CD47 polypeptide, in whole or in part, with a second non-human animal, said second non-human animal having a genome comprising a SIRPa gene that encodes a SIRPa polypeptide comprising an extracellular portion of a human SIRPa polypeptide (e.g., amino acids
  • the present invention provides a method of providing a non- human animal whose genome comprises a CD47 gene that encodes an extracellular portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous CD47 polypeptide, the method comprising modifying the genome of a non-human animal so that it comprises a CD47 gene that encodes the extracellular portion of a human CD47 polypeptide linked to the intracellular portion of an endogenous CD47 polypeptide thereby providing said non-human animal.
  • a CD47 gene encodes a CD47 polypeptide that comprises an extracellular portion and a transmembrane portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous non-human CD47 polypeptide. In other embodiments, a CD47 gene encodes a CD47 polypeptide that comprises an extracellular portion of a human CD47 polypeptide linked to a
  • transmembrane portion and an intracellular portion of an endogenous non-human CD47 polypeptide are transmembrane portion and an intracellular portion of an endogenous non-human CD47 polypeptide.
  • the modifying the genome of a non-human animal is performed in a non-human embryonic stem cell.
  • the non- human embryonic stem cell is a rodent embryonic stem cell; in some embodiments, a mouse embryonic stem cell; in some embodiments, a rat embryonic stem cell.
  • the method further comprises modifying the genome of the non-human animal so that it comprises a SIRPa gene that encodes the extracellular portion of a human SIRPa polypeptide (e.g., amino acids corresponding to residues 28-362 of a human SIRPa polypeptide) linked to the intracellular portion of an endogenous SIRPa polypeptide.
  • a human SIRPa polypeptide e.g., amino acids corresponding to residues 28-362 of a human SIRPa polypeptide
  • the modifying the genome of the non-human animal so that it comprises a SIRPa gene that encodes the extracellular portion of a human SIRPa polypeptide (e.g., amino acids corresponding to residues 28-362 of a human SIRPa polypeptide) linked to the intracellular portion of an endogenous SIRPa polypeptide is performed prior to modifying the genome of the non-human animal so that it comprises a CD47 gene that encodes the extracellular portion and optionally a transmembrane portion of a human CD47 polypeptide linked to the intracellular portion of an endogenous CD47 polypeptide.
  • the method further comprises breeding a non-human animal whose genome comprises a CD47 gene that encodes an extracellular portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous CD47 polypeptide with a second non-human animal, said second non-human animal having a genome comprising a SIRPa gene that encodes a SIRPa polypeptide comprising an extracellular portion of a human SIRPa polypeptide (e.g., amino acids corresponding to residues 28-362 of a human SIRPa polypeptide) and an intracellular portion of an endogenous SIRPa polypeptide.
  • the present invention provides a non-human animal obtainable by methods as described herein.
  • the present invention provides a method of engrafting human cells into a non-human animal, the method comprising steps of providing a non- human animal whose genome comprises a CD47 gene that encodes the extracellular portion of a human CD47 polypeptide linked to the intracellular portion of an endogenous CD47 polypeptide; and transplanting one or more human cells into said non-human animal.
  • the method further comprises a step of assaying engraftment of the one or more human cells in said non-human animal.
  • a step of assaying comprises comparing the engraftment of the one or more human cells to the engraftment in one or more wild-type non-human animals or in one or more non-human animals whose genome does not comprise a CD47 gene that encodes the extracellular portion of a human CD47 polypeptide linked to the intracellular portion of an endogenous CD47 polypeptide.
  • human cells are hematopoietic stem cells. In some embodiments, human cells are transplanted intravenously. In some embodiments, human cells are transplanted intraperitoneally. In some embodiments, human cells are
  • the present invention provides a method of assessing the therapeutic efficacy of a drug targeting human cells, the method comprising providing a non-human animal whose genome comprises a CD47 gene that encodes an extracellular portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous CD47 polypeptide; transplanting one or more human cells into said non-human animal; administering a drug candidate to said non-human animal; and monitoring the human cells in the non-human animal to determine the therapeutic efficacy of the drug candidate.
  • human cells are cancer cells and the drug candidate is an anti-cancer drug candidate.
  • a drug candidate is an antibody.
  • a non-human animal further comprises human immune cells.
  • a drug candidate is a bi-specific antibody that binds human CD47 and an antigen on transplanted human cancer cells.
  • the present invention provides a method comprising providing one or more cells whose genome includes a CD47 gene that encodes an extracellular portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous CD47 polypeptide; incubating the one or more cells with a labeled substrate; and measuring phagocytosis of the labeled substrate by the one or more cells.
  • the substrate is fluorescently labeled.
  • the substrate is labeled with an antibody.
  • the substrate is one or more red blood cells.
  • the substrate is one or more bacterial cells.
  • the substrate is one or more tumor cells.
  • the present invention provides a method comprising providing a non-human animal whose genome includes a CD47 gene that encodes an extracellular portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous CD47 polypeptide; exposing the non-human animal to an antigen; and measuring phagocytosis of the antigen by one or more cells of the non-human animal.
  • the step of exposing comprises exposing the non-human animal to an antigen that is fluorescently labeled.
  • the step of exposing comprises exposing the non-human animal to one or more cells that comprise the antigen.
  • the step of exposing comprises exposing the non-human animal to one or more human cells comprising the antigen or to one or more bacterial cells comprising the antigen. In some embodiments, the step of exposing comprises exposing the non-human animal to one or more cells that have been transformed with the antigen so that the antigen is expressed on the surface of the one or more transformed cells. In some embodiments, the step of exposing comprises exposing the non-human animal to one or more tumor cells that comprise the antigen.
  • the present invention provides methods for identification or validation of a drug or vaccine, the method comprising the steps of delivering a drug or vaccine to a non-human animal whose genome includes a CD47 gene that encodes an extracellular portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous CD47 polypeptide, and monitoring one or more of the immune response to the drug or vaccine, the safety profile of the drug or vaccine, or the effect on a disease or condition.
  • monitoring the safety profile includes determining if the non-human animal exhibits a side effect or adverse reaction as a result of delivering the drug or vaccine.
  • a side effect or adverse reaction is selected from morbidity, mortality, alteration in body weight, alteration of the level of one or more enzymes (e.g., liver), alteration in the weight of one or more organs, loss of function (e.g., sensory, motor, organ, etc.), increased susceptibility to one or more diseases, alterations to the genome of the non-human animal, increase or decrease in food consumption and complications of one or more diseases.
  • one or more enzymes e.g., liver
  • loss of function e.g., sensory, motor, organ, etc.
  • increased susceptibility to one or more diseases alterations to the genome of the non-human animal, increase or decrease in food consumption and complications of one or more diseases.
  • the present invention provides use of a non-human animal as described herein in the development of a drug or vaccine for use in medicine, such as use as a medicament.
  • the present invention provides use of a non-human animal as described herein in the manufacture of a medicament for the treatment of cancer or a neoplasm.
  • the present invention provides use of a non-human animal as described herein to assess the efficacy of a therapeutic drug targeting human cells.
  • a non-human animal of the present invention is transplanted with human cells and a drug candidate targeting said human cells is administered to the animal.
  • efficacy of the drug is determined by monitoring the human cells in the non-human animal after the administration of the drug.
  • the present invention provides a non-human animal or cell as described herein for use in the development and/or identification of a drug (e.g., an antibody) for therapy or diagnosis.
  • the present invention provides a non-human animal or cell as described herein for use in the development and/or identification of a drug (e.g., an antibody) for the treatment, prevention or amelioration of cancer or a neoplasm.
  • a drug e.g., an antibody
  • the present invention provides a method of assessing the pharmacokinetics of a drug targeting human CD47, the method comprising the steps of administering the drug to a non-human animal as described herein, and performing an assay to determine one or more pharmacokinetic properties of the drug targeting human CD47.
  • the present invention provides a method of assessing the on- target toxicity of a drug targeting human CD47, the method comprising the steps of administering the drug to a non-human animal as described herein, and performing an assay for one or more parameters associated with on-target toxicity of a drug.
  • the present invention provides a method of assessing the off-target toxicity of a drug targeting human CD47, the method comprising the steps of administering the drug to a non-human animal as described herein, and performing an assay for one or more parameters associated with off-target toxicity of a drug.
  • a non-human animal as described herein is a rodent whose genome includes a CD47 gene that encodes an extracellular portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous CD47 polypeptide; in some embodiments, a rodent is a mouse; in some embodiments, a rodent is a rat.
  • a drug targeting human CD47 is a CD47 antagonist. In some certain embodiments, a CD47 antagonsit is an anti-CD47 antibody. In some embodiments, a drug targeting human CD47 is a CD47 agonist.
  • a CD47 gene of the present invention includes a CD47 gene as described herein.
  • a CD47 polypeptide of the present invention includes a CD47 polypeptide as described herein.
  • a non-human animal of the present invention does not detectably express a full-length endogenous non-human CD47 polypeptide.
  • a non-human animal of the present invention does not detectably express an extracellular portion of an endogenous CD47 polypeptide.
  • a non- human animal of the present invention does not detectably express an extracellular portion of both an endogenous CD47 polypeptide and an endogenous SIRPa polypeptide.
  • an extracellular portion of a human CD47 polypeptide comprises amino acids corresponding to residues 19-141 of a human CD47 polypeptide as described herein.
  • an N-terminal immunoglobulin V domain of a human CD47 polypeptide comprises amino acids corresponding to residues 19-127 of a human CD47 polypeptide as described herein.
  • non-human animals, cells, tissues, embryonic stem cells and/or embryos of the present invention have a genome that further comprises a SIRPa gene that encodes a SIRPa polypeptide comprising an extracellular portion of a human SIRPa polypeptide (e.g., amino acids corresponding to residues 28-362 of a human SIRPa polypeptide) and an intracellular portion of an endogenous SIRPa polypeptide.
  • a SIRPa gene that encodes a SIRPa polypeptide comprising an extracellular portion of a human SIRPa polypeptide (e.g., amino acids corresponding to residues 28-362 of a human SIRPa polypeptide) and an intracellular portion of an endogenous SIRPa polypeptide.
  • a non-human animal of the present invention is a rodent; in some embodiments, a mouse; in some embodiments, a rat.
  • Figure 1 shows a diagram, not to scale, of the genomic organization of a non- human (e.g., mouse) and human Cluster of Differentiation 47 (CD47) genes. Exons are numbered beneath each exon.
  • a non- human e.g., mouse
  • CD47 human Cluster of Differentiation 47
  • Figure 2 shows a diagram, not to scale, of an exemplary method for humanization of a non-human Cluster of Differentiation 47 (CD47) gene.
  • CD47 Cluster of Differentiation 47
  • Figure 3 shows a diagram, not to scale, of the genomic orgainzation of a mouse and human Cluster of Differentiation 47 (CD47) genes. Locations of probes used in an assay described in Example 1 are indicated.
  • Figure 4 shows an exemplary histogram of CD47 expression in red blood cells from humanized CD47 mice detected by anti-CD47 antibodies.
  • Ab A, Ab B, Ab C, Ab D and Ab E anti-CD47 antibodies;
  • hIgG4s human IgG4 of irrelevant specificity with modified Fc region that has reduced effector function;
  • hIgG4 human IgG4 antibody of irrelevant specificity.
  • WT wild-type
  • HuCD47 humanized CD47
  • Ab A, Ab B, Ab C, Ab D and Ab E anti-CD47 antibodies
  • hIgG4s human IgG4 of irrelevant specificity with modified Fc region that has reduced effector function
  • hIgG4 human IgG4 antibody of irrelevant specificity.
  • Figure 6 shows exemplary pharmacokinetic profiles of anti-CD47 antibodies in humanized CD47 mice represented as antibody concentration (in ⁇ g/mL, y-axis) over time (in days, x-axis); Ab F, Ab G, Ab H and Ab I: anti-CD47 antibodies; hIgG4s: human IgG4 antibody of irrelevant specificity with modified Fc region that has reduced effector function.
  • Figure 7 shows exemplary pharmacokinetic profiles of anti-CD47 antibodies in humanized CD47/SIRPa mice (CD47 hu/hu SIRPa hu/hu ) represented as antibody
  • Figure 8 shows exemplary pharmacokinetic profiles of anti-CD47 antibodies in humanized CD47/SIRPa mice (CD47 hu/hu SIRPa hu/hu ) represented as antibody
  • Ab J Ab F: anti-CD47 antibodies
  • Ab Fs Ab F with modified Fc region that has reduced effector function
  • Ab Fmono monovalent version of Ab F
  • hIgG4s human IgG4 antibody of irrelevant specificity with modified Fc region that has reduced effector function.
  • Figure 9 shows exemplary pharmacokinetic profiles of anti-CD47 antibodies in wild type mice represented as antibody concentration (in mcg/mL, y-axis) over time (in days, x-axis). Mice demonstrating mouse anti -human antibody response (MAFIA) were excluded.
  • Ab J, Ab F, Ab I and Ab G anti-CD47 antibodies
  • hIgG4s human IgG4 antibody of irrelevant specificity with modified Fc region that has reduced effector function (star: all points after 15 days were excluded from hIgG4s treatment group due to MAHA);
  • Ab J F(ab') 2 F(ab') 2 fragment of Ab J.
  • the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
  • biologically active refers to a characteristic of any agent that has activity in a biological system, in vitro or in vivo (e.g., in an organism). For instance, an agent that, when present in an organism, has a biological effect within that organism, is considered to be biologically active.
  • an agent that, when present in an organism, has a biological effect within that organism is considered to be biologically active.
  • a portion of that protein or polypeptide that shares at least one biological activity of the protein or polypeptide is typically referred to as a "biologically active" portion.
  • conservative amino acid substitution refers to substitution of an amino acid residue by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or
  • a conservative amino acid substitution will not substantially change the functional properties of interest of a protein, for example, the ability of a receptor to bind to a ligand.
  • groups of amino acids that have side chains with similar chemical properties include: aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxyl side chains such as serine and threonine; amide- containing side chains such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, arginine, and histidine; acidic side chains such as aspartic acid and glutamic acid; and, sulfur-containing side chains such as cysteine and methionine.
  • Conservative amino acids substitution groups include, for example, valine/leucine/isoleucine, phenylalanine/tyrosine, lysine/arginine, alanine/valine, glutamate/aspartate, and asparagine/glutamine.
  • a conservative amino acid substitution can be a substitution of any native residue in a protein with alanine, as used in, for example, alanine scanning mutagenesis.
  • a conservative substitution is made that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al., (1992) Exhaustive Matching of the Entire Protein Sequence Database, Science 256:1443-45, hereby incorporated by reference.
  • the substitution is a moderately conservative substitution wherein the substitution has a nonnegative value in the PAM250 log-likelihood matrix.
  • a control is a reaction or assay that is performed simultaneously with a test reaction or assay to provide a comparator.
  • a "controF may refer to a "control animaF.
  • a "control animaF may have a modification as described herein, a modification that is different as described herein, or no modification (i.e., a wild-type animal).
  • a control is a historical control (i.e., of a test or assay performed previously, or an amount or result that is previously known).
  • a control is or comprises a printed or otherwise saved record.
  • a control may be a positive control or a negative control.
  • disruption refers to the result of a homologous recombination event with a DNA molecule (e.g., with an endogenous homologous sequence such as a gene or gene locus.
  • a disruption may achieve or represent an insertion, deletion, substitution, replacement, missense mutation, or a frame- shift of a DNA sequence(s), or any combination thereof.
  • Insertions may include the insertion of entire genes or fragments of genes, e.g., exons, which may be of an origin other than the endogenous sequence (e.g., a heterologous sequence).
  • exons which may be of an origin other than the endogenous sequence (e.g., a heterologous sequence).
  • a disruption may increase expression and/or activity of a gene or gene product (e.g., of a protein encoded by a gene). In some embodiments, a disruption may decrease expression and/or activity of a gene or gene product. In some embodiments, a disruption may alter sequence of a gene or an encoded gene product (e.g., an encoded protein). In some embodiments, a disruption may truncate or fragment a gene or an encoded gene product (e.g., an encoded protein). In some embodiments, a disruption may extend a gene or an encoded gene product; in some such embodiments, a disruption may achieve assembly of a fusion protein. In some embodiments, a disruption may affect level but not activity of a gene or gene product.
  • a disruption may affect activity but not level of a gene or gene product. In some embodiments, a disruption may have no significant effect on level of a gene or gene product. In some embodiments, a disruption may have no significant effect on activity of a gene or gene product. In some embodiments, a disruption may have no significant effect on either level or activity of a gene or gene product.
  • determining means determining if an element is present or not. These terms include both quantitative and/or qualitative determinations. Assaying may be relative or absolute. "Assaying for the presence of can be determining the amount of something present and/or determining whether or not it is present or absent.
  • endogenous locus or "endogenous gene”, as used herein, refers to a genetic locus found in a parent or reference organism prior to introduction of a disruption, deletion, replacement, alteration, or modification as described herein.
  • the endogenous locus has a sequence found in nature.
  • the endogenous locus is a wild type locus.
  • the reference organism is a wild-type organism.
  • the reference organism is an engineered organism.
  • the reference organism is a laboratory- bred organism (whether wild-type or engineered).
  • endogenous promoter refers to a promoter that is naturally associated, e.g., in a wild-type organism, with an endogenous gene.
  • heterologous refers to an agent or entity from a different source.
  • the term clarifies that the relevant polypeptide, gene, or gene product: 1) was engineered by the hand of man; 2) was introduced into the cell or organism (or a precursor thereof) through the hand of man (e.g., via genetic engineering); and/or 3) is not naturally produced by or present in the relevant cell or organism (e.g., the relevant cell type or organism type).
  • a host cell refers to a cell into which a heterologous (e.g., exogenous) nucleic acid or protein has been introduced. Persons of skill upon reading this disclosure will understand that such terms refer not only to the particular subject cell, but also is used to refer to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell” as used herein.
  • a host cell is or comprises a prokaryotic or eukaryotic cell.
  • a host cell is any cell that is suitable for receiving and/or producing a heterologous nucleic acid or protein, regardless of the Kingdom of life to which the cell is designated.
  • Exemplary cells include those of prokaryotes and eukaryotes (single-cell or multiple-cell), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P.
  • the cell is a human, monkey, ape, hamster, rat, or mouse cell.
  • the cell is eukaryotic and is selected from the following cells: CHO (e.g., CHO Kl, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell, Vero, CVl, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV- 1, U937, 3T3, L cell, C127 cell, SP2/0, NS-0, MMT 060562, Sertoli cell, BRL 3 A cell, HT1080 cell, myeloma cell, tumor cell, and a cell line derived from an aforementioned cell.
  • CHO e.g., CHO Kl, DXB-11 CHO, Veggie-CHO
  • COS e
  • the cell comprises one or more viral genes, e.g., a retinal cell that expresses a viral gene (e.g., a PER.C6TM cell).
  • a host cell is or comprises an isolated cell.
  • a host cell is part of a tissue.
  • a host cell is part of an organism.
  • humanized is used herein in accordance with its art-understood meaning to refer to nucleic acids or proteins whose structures (i.e., nucleotide or amino acid sequences) include portions that correspond substantially or identically with structures of a particular gene or protein found in nature in a non-human animal, and also include portions that differ from that found in the relevant particular non-human gene or protein and instead correspond more closely with comparable structures found in a corresponding human gene or protein.
  • a "humanized” gene is one that encodes a polypeptide having substantially the amino acid sequence as that of a human polypeptide (e.g., a human protein or portion thereof - e.g., characteristic portion thereof).
  • a "humanized" gene may encode a polypeptide having an extracellular portion having an amino acid sequence as that of a human extracellular portion and the remaining sequence as that of a non-human (e.g., mouse) polypeptide.
  • a humanized gene comprises at least a portion of a DNA sequence of a human gene.
  • a humanized gene comprises an entire DNA sequence of a human gene.
  • a humanized protein comprises a sequence having a portion that appears in a human protein.
  • a humanized protein comprises an entire sequence of a human protein and is expressed from an endogenous locus of a non-human animal that corresponds to the homolog or ortholog of the human gene.
  • identity refers to identity as determined by a number of different algorithms known in the art that can be used to measure nucleotide and/or amino acid sequence identity.
  • identities as described herein are determined using a ClustalW v. 1.83 (slow) alignment employing an open gap penalty of 10.0, an extend gap penalty of 0.1, and using a Gonnet similarity matrix (MACVECTORTM 10.0.2, Mac Vector Inc., 2008).
  • isolated refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) designed, produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated.
  • isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
  • a substance is "pure” if it is substantially free of other components.
  • a substance may still be considered “isolated” or even “pure”, after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients.
  • a biological polymer such as a polypeptide or polynucleotide that occurs in nature is considered to be "isolated” when: a) by virtue of its origin or source of derivation is not associated with some or all of the components that accompany it in its native state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in nature; or c) is expressed by or is otherwise in association with components from a cell or other expression system that is not of the species that produces it in nature.
  • a polypeptide that is chemically synthesized or is synthesized in a cellular system different from that which produces it in nature is considered to be an "isolated” polypeptide.
  • a polypeptide that has been subjected to one or more purification techniques may be considered to be an "isolated" polypeptide to the extent that it has been separated from other components: a) with which it is associated in nature; and/or b) with which it was associated when initially produced.
  • non-human animal refers to any vertebrate organism that is not a human.
  • a non-human animal is acyclostome, a bony fish, a cartilaginous fish (e.g., a shark or a ray), an amphibian, a reptile, a mammal, or a bird.
  • a non-human mammal is a primate, a goat, a sheep, a pig, a dog, a cow, or a rodent.
  • a non-human animal is a rodent such as a rat or a mouse.
  • nucleic acid refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain.
  • a “nucleic acid” is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage.
  • nucleic acid refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides); in some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising individual nucleic acid residues.
  • a "nucleic acid” is or comprises RNA; in some embodiments, a “nucleic acid” is or comprises DNA. In some embodiments, a “nucleic acid” is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a “nucleic acid” is, comprises, or consists of one or more nucleic acid analogs. In some
  • a nucleic acid analog differs from a "nucleic acid” in that it does not utilize a phosphodiester backbone.
  • a "nucleic acid” is, comprises, or consists of one or more "peptide nucleic acids", which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention.
  • a "nucleic acid” has one or more phosphorothioate and/or 5'-N- phosphoramidite linkages rather than phosphodiester bonds.
  • a "nucleic acid” is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine).
  • nucleosides e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine.
  • a "nucleic acid” is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5- fluorouridine, C5-iodouridine, C 5 -propynyl-uridine, C5-propynyl-cytidine, C5- methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, inter
  • a "nucleic acid” comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids.
  • a "nucleic acid' has a nucleotide sequence that encodes a functional gene product such as an RNA or protein.
  • a "nucleic acid' includes one or more introns.
  • a "nucleic acid' is prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis.
  • a "nucleic acid' is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long.
  • a "nucleic acid' is single stranded; in some embodiments, a "nucleic acid' is double stranded.
  • a "nucleic acid' has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a "nucleic acid' has enzymatic activity.
  • operably linked 1 refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • a control sequence "operably linked 1 to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • operably linked 1 sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
  • expression control sequence refers to polynucleotide sequences, which are necessary to effect the expression and processing of coding sequences to which they are ligated.
  • “Expression control sequences” include: appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion.
  • the nature of such control sequences differs depending upon the host organism. For example, in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence, while in eukaryotes, typically, such control sequences include promoters and transcription termination sequence.
  • control sequences is intended to include components whose presence is essential for expression and processing, and can also include additional components whose presence is
  • leader sequences and fusion partner sequences.
  • polypeptide refers to any polymeric chain of amino acids.
  • a polypeptide has an amino acid sequence that occurs in nature.
  • a polypeptide has an amino acid sequence that does not occur in nature.
  • a polypeptide has an amino acid sequence that contains portions that occur in nature separately from one another (i.e., from two or more different organisms, for example, human and non-human portions).
  • a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man.
  • recombinant is intended to refer to polypeptides (e.g., CD47polypeptides as described herein) that are designed, engineered, prepared, expressed, created or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell, polypeptides isolated from a recombinant, combinatorial human polypeptide library (Hoogenboom H. R., (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W. E., (2002) Clin. Biochem. 35:425-445; Gavilondo J. V., and Larrick J. W.
  • polypeptides e.g., CD47polypeptides as described herein
  • recombinant means such as polypeptides expressed using a recombinant expression vector transfected into a host cell, polypeptides isolated from a recombinant, combinatorial human polypeptide library (Hoogenboom H
  • one or more of such selected sequence elements is found in nature.
  • one or more of such selected sequence elements is designed in silico.
  • one or more such selected sequence elements result from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source.
  • a recombinant polypeptide is comprised of sequences found in the genome of a source organism of interest (e.g., human, mouse, etc.).
  • a recombinant polypeptide has an amino acid sequence that resulted from mutagenesis (e.g., in vitro or in vivo, for example in a non-human animal), so that the amino acid sequences of the recombinant polypeptides are sequences that, while originating from and related to polypeptides sequences, may not naturally exist within the genome of a non-human animal in vivo.
  • replacement is used herein to refer to a process through which a "replaced" nucleic acid sequence (e.g., a gene) found in a host locus (e.g., in a genome) is removed from that locus, and a different, "replacement" nucleic acid is located in its place.
  • the replaced nucleic acid sequence and the replacement nucleic acid sequences are comparable to one another in that, for example, they are homologous to one another and/or contain corresponding elements (e.g., protein-coding elements, regulatory elements, etc.).
  • a replaced nucleic acid sequence includes one or more of a promoter, an enhancer, a splice donor site, a splice receiver site, an intron, an exon, an untranslated region (UTR); in some embodiments, a replacement nucleic acid sequence includes one or more coding sequences. In some embodiments, a replacement nucleic acid sequence is a homolog of the replaced nucleic acid sequence. In some embodiments, a replacement nucleic acid sequence is an ortholog of the replaced sequence. In some embodiments, a replacement nucleic acid sequence is or comprises a human nucleic acid sequence.
  • the replaced nucleic acid sequence is or comprises a human nucleic acid sequence
  • the replaced nucleic acid sequence is or comprises a rodent sequence (e.g., a mouse or rat sequence).
  • the nucleic acid sequence so placed may include one or more regulatory sequences that are part of source nucleic acid sequence used to obtain the sequence so placed (e.g., promoters, enhancers, 5'- or 3 '-untranslated regions, etc.).
  • the replacement is a substitution of an endogenous sequence with a heterologous sequence that results in the production of a gene product from the nucleic acid sequence so placed (comprising the heterologous sequence), but not expression of the endogenous sequence; the replacement is of an endogenous genomic sequence with a nucleic acid sequence that encodes a protein that has a similar function as a protein encoded by the endogenous sequence (e.g., the endogenous genomic sequence encodes a CD47 protein, and the DNA fragment encodes one or more human CD47 proteins).
  • an endogenous gene or fragment thereof is replaced with a corresponding human gene or fragment thereof.
  • a corresponding human gene or fragment thereof is a human gene or fragment that is an ortholog of, or is substantially similar or the same in structure and/or function, as the endogenous gene or fragment thereof that is replaced.
  • CD47 protein refers to a multi-pass transmembrane protein that belongs to the immunoglobulin superfamily and has an extracellular amino-terminal immunoglobulin V domain, five transmembrane domains and a short carboxyl-terminal intracellular tail.
  • CD47 is expressed on the cell surface and is involved in interactions between membrane surface proteins such as, for example, integrins, SIRPa and thrombospondin-1 (TSP-1).
  • CD47 is expressed in normal tissues and up-regulated in many human cancers. CD47 has been shown to be involved in several cellular processes such as, for example, apoptosis, proliferation, adhesion and migration.
  • CD47 -expressing cell refers to a cell that expresses a CD47 transmembrane protein. In some embodiments, a CD47-expressing cell expresses a CD47 transmembrane protein on its surface.
  • a CD47 protein expressed on the surface of the cell in an amount sufficient to mediate cell-to-cell interactions via the CD47 transmembrane protein expressed on the surface of the cell.
  • exemplary CD47- expressing cells include neurons, immune cells, keratinocytes, and circulating cells.
  • CD47-expressing cells regulate the interaction of immune cells and circulating cells to regulate various cellular processes such as adhesion, cell proliferation and/or apoptosis, angiogenesis and inflammation.
  • non-human animals of the present invention demonstrate regulation of various cellular processes (as described herein) via humanized CD47 proteins expressed on the surface of one more cells of the non-human animal.
  • a reference agent is used herein to describe a standard or control agent, cohort, individual, population, sample, sequence or value against which an agent, animal, cohort, individual, population, sample, sequence or value of interest is compared.
  • a reference agent, cohort, individual, population, sample, sequence or value is tested and/or determined substantially simultaneously with the testing or determination of the agent, cohort, individual, population, sample, sequence or value of interest.
  • a reference agent, cohort, individual, population, sample, sequence or value is a historical reference, optionally embodied in a tangible medium.
  • a reference may refer to a control.
  • a “reference” may refer to a "reference animaF.
  • a “reference animal” may have a modification as described herein, a modification that is different as described herein or no modification (i.e., a wild- type animal).
  • a reference agent, animal, cohort, individual, population, sample, sequence or value is determined or characterized under conditions comparable to those utilized to determine or characterize the agent, animal (e.g., a mammal), cohort, individual, population, sample, sequence or value of interest.
  • substantially homology refers to a comparison between amino acid or nucleic acid sequences. As will be appreciated by those of ordinary skill in the art, two sequences are generally considered to be “substantially homologous” if they contain homologous residues in corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues will appropriately similar structural and/or functional characteristics. For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as “hydrophobic” or “hydrophilic” amino acids, and/or as having "polar” or “non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a "homologous" substitution. Typical amino acid categorizations are summarized in Table 1 and 2.
  • amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences.
  • Exemplary such programs are described in Altschul et al., (1990) Basic local alignment search tool, J. Mol. Biol, 215(3): 403-410; Altschul et al., (1997) Methods in Enzymology; Altschul et al, "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res.
  • two sequences are considered to be substantially homologous if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are homologous over a relevant stretch of residues.
  • the relevant stretch is a complete sequence. In some embodiments, the relevant stretch is at least 9, 10, 11, 12, 13, 14, 15, 16, 17 or more residues. In some embodiments, the relevant stretch includes contiguous residues along a complete sequence. In some embodiments, the relevant stretch includes discontinuous residues along a complete sequence. In some embodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more residues.
  • amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul et al., (1990) Basic local alignment search tool, J. Mol. Biol, 215(3): 403-410; Altschul et al., Methods in
  • two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over a relevant stretch of residues.
  • the relevant stretch is a complete sequence. In some embodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more residues.
  • targeting vector refers to a polynucleotide molecule that comprises a targeting region.
  • a targeting region comprises a sequence that is identical or substantially identical to a sequence in a target cell, tissue or animal and provides for integration of the targeting construct into a position within the genome of the cell, tissue or animal via homologous recombination.
  • Targeting regions that target using site-specific recombinase recognition sites are also included.
  • a targeting construct of the present invention further comprises a nucleic acid sequence or gene of particular interest, a selectable marker, control and or regulatory sequences, and other nucleic acid sequences that allow for recombination mediated through exogenous addition of proteins that aid in or facilitate recombination involving such sequences.
  • a targeting construct of the present invention further comprises a gene of interest in whole or in part, wherein the gene of interest is a heterologous gene that encodes a protein, in whole or in part, that has a similar function as a protein encoded by an endogenous sequence.
  • a targeting construct of the present invention further comprises a humanized gene of interest, in whole or in part, wherein the humanized gene of interest encodes a protein, in whole or in part, that has a similar function as a protein encoded by the endogenous sequence.
  • variable refers to an entity that shows significant structural identity with a reference entity, but differs structurally from the reference entity in the presence or level of one or more chemical moieties as compared with the reference entity. In many embodiments, a “variant” also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a “variant” of a reference entity is based on its degree of structural identity with the reference entity. As will be appreciated by those skilled in the art, any biological or chemical reference entity has certain characteristic structural elements. A “variant”, by definition, is a distinct chemical entity that shares one or more such characteristic structural elements.
  • a small molecule may have a characteristic core structural element (e.g., a macrocycle core) and/or one or more characteristic pendent moieties so that a variant of the small molecule is one that shares the core structural element and the characteristic pendent moieties but differs in other pendent moieties and/or in types of bonds present (single vs. double, E vs.
  • a characteristic core structural element e.g., a macrocycle core
  • characteristic pendent moieties e.g., one or more characteristic pendent moieties
  • a polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and/or contributing to a particular biological function
  • a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to on another in linear or three-dimensional space.
  • a "variant polypeptide" may differ from a reference polypeptide as a result of one or more differences in amino acid sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone.
  • a "variant polypeptide” shows an overall sequence identity with a reference polypeptide that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%.
  • a "variant polypeptide” does not share at least one characteristic sequence element with a reference polypeptide.
  • the reference polypeptide has one or more biological activities.
  • a "variant polypeptide” shares one or more of the biological activities of the reference polypeptide.
  • a "variant polypeptide” lacks one or more of the biological activities of the reference polypeptide.
  • a "variant polypeptide” shows a reduced level of one or more biological activities as compared with the reference polypeptide.
  • a polypeptide of interest is considered to be a "variant" of a parent or reference polypeptide if the polypeptide of interest has an amino acid sequence that is identical to that of the parent but for a small number of sequence alterations at particular positions. Typically, fewer than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of the residues in the variant are substituted as compared with the parent.
  • a "variant" has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue as compared with a parent.
  • a "variant” has a very small number (e.g., fewer than 5, 4, 3, 2, or 1) number of substituted functional residues (i.e., residues that participate in a particular biological activity). Furthermore, a "variant” typically has not more than 5, 4, 3, 2, or 1 additions or deletions, and often has no additions or deletions, as compared with the parent. Moreover, any additions or deletions are typically fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly are fewer than about 5, about 4, about 3, or about 2 residues. In some embodiments, the parent or reference polypeptide is one found in nature.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is associated.
  • vectors are capable of extra-chromosomal replication and/or expression of nucleic acids to which they are linked in a host cell such as a eukaryotic and/or prokaryotic cell.
  • vectors capable of directing the expression of operatively linked genes are referred to herein as "expression vectors.”
  • wild-type has its art-understood meaning that refers to an entity having a structure and/or activity as found in nature in a "normal” (as contrasted with mutant, diseased, altered, etc.) state or context.
  • wild- type genes and polypeptides often exist in multiple different forms (e.g., alleles).
  • the present invention provides, among other things, improved and/or engineered non-human animals having humanized genetic material encoding a cluster of
  • CD47 differentiation 47 gene for determining the therapeutic efficacy of CD47 antagonists (e.g., an anti-CD47 antibody) for the treatment of cancer, and assays in transplant engraftment, activation and phagocytosis and signal transduction. It is contemplated that such non-human animals provide an improvement in determining the therapeutic efficacy of CD47 antagonists and their potential for CD47 blockade. It is also contemplated that such non-human animals provide an improvement in transplant engraftment of human cells. Therefore, the present invention is particularly useful for the development of anti-CD47 therapies for the treatment of various cancers, as well as for maintaining human hematopoietic cells in non-human animals.
  • CD47 antagonists e.g., an anti-CD47 antibody
  • the present invention encompasses the humanization of a murine CD47 gene resulting in expression of a humanized CD47 protein on the surface of cells of the non-human animal.
  • humanized CD47 proteins have the capacity to provide a source of human CD47 + cells for determining the efficacy of anti-CD47 therapeutics to activate phagocytosis of tumor cells.
  • humanized CD47 proteins have the capacity to recognize engrafted human cells via engagement of other cell surface proteins and ligands present on the surface of the engrafted human cells (e.g., SIRPa).
  • non-human animals of the present invention are capable of activating phagocytosis via blockade of CD47 signaling through the humanized CD47 protein expressed on the surface of cells of the non-human animal.
  • non-human animals of the present invention are capable of receiving transplanted human hematopoietic cells; in some embodiments, such non-human mammals develop and/or have an immune system comprising human cells.
  • humanized CD47 proteins have a sequence corresponding to the N-terminal immunoglobulin V domain of a human CD47 protein.
  • humanized CD47 proteins have a sequence corresponding to an N-terminal portion of a human CD47 protein that comprises an extracellular portion and a transmembrane portion of a human CD47 protein, wherein the extracellular portion includes the N-terminal immunoglobulin V domain of the human CD47 protein and the transmembrane portion includes the five transmembrane domains of the human CD47 protein.
  • humanized CD47 proteins have a sequence corresponding to the intracytoplasmic tail of a non-human (e.g., murine) CD47 protein.
  • humanized CD47 proteins have a sequence corresponding to amino acid residues 19-292 (or 19-141, or 19-127) of a human CD47 protein.
  • non-human animals of the present invention comprise an endogenous CD47 gene that contains genetic material from the non-human animal and a heterologous species (e.g., a human).
  • non-human animals of the present invention comprise a humanized CD47 gene, wherein the humanized CD47 gene comprises exons of a human CD47 gene that encode an
  • the humanized CD47 gene comprises human CD47 exons, e.g., exons 2-7, encoding an N-terminal portion of a human CD47 protein that comprises an extracellular portion and a transmembrane portion of a human CD47 protein, wherein the extracellular portion includes the N-terminal immunoglobulin V domain of the human CD47 protein and the transmembrane portion includes the five transmembrane domains of the human CD47 protein.
  • the humanized CD47 gene comprises non-human CD47 exons that encode the signal peptide, in whole or in part, and the intracytoplasmic tail of a non-human CD47 protein.
  • the humanized CD47 gene comprises non-human CD47 exon 1 and exon(s) downstream of exon 7 that encode the intracytoplasmic tail and the 3' UTR.
  • isoform 2 of both mouse and human CD47 shown in Table 3 have two exons downstream of exon 7, designated as exon 8 and 9.
  • CD47 originally named integrin-associated protein (IAP) for its role in signal transduction from integrins on immune cells, is a transmembrane protein that includes an N-terminal immunoglobulin V (IgV) domain, five transmembrane domains, and a short C- terminal intracytoplasmic tail.
  • the intracytoplasmic tail differs in length according to four alternatively spliced isoforms that have been identified.
  • CD47 (or IAP) was initially described as being expressed on all tissues (isoform 2), neurons (isoform 4) and keratinocytes and macrophages (isoform 1; see Reinhold et al., (1995) J. Cell Sci.
  • CD47 is known to interact with several other cell surface proteins such as, for example,
  • CD47 interacts with SIRPa and leads to bidirectional signaling that regulates a variety of cell-to-cell responses such as, for example, inhibition of phagocytosis and T cell activation.
  • CD47- SIRPa interaction has come into focus in recent years for its role in providing tumor cells with the capacity to evade immune surveillance.
  • CD47 binding to SIRPa normally provides protection through anti-phagocytic signals ("don't eat me") for normal cells.
  • tumors also express anti-phagocytic signals, including CD47, to evade destruction by phagocytosis.
  • CD47 is known to be upregulated in several hematologic cancers and contribute to both the growth and dissemination of tumors (Chao et al., (2012) Curr Opin Immunol.24(2): 225-232).
  • Exemplary CD47 sequences for mouse and human are set forth in Table 3.
  • signal peptides are underlined, extracellular sequences are bold font and intracytoplasmic sequences are italicized.
  • humanized protein sequences non-human sequences are indicated in regular font, human sequences are indicated in bold font, and signal peptides are underlined.
  • the isoforms differ in the number of exons. For example, isoforms 1-4 of the human CD47 gene have a total of 8, 9, 10 and 11 exons, respectively, with exons 2-7 of each isoform encoding the extracellular domain and the five transmembrane domains.
  • Non-human animals are provided that express humanized CD47 proteins on the surface of cells of the non-human animals resulting from a genetic modification of an endogenous locus of the non-human animal that encodes a CD47 protein.
  • Suitable examples described herein include rodents, in particular, mice.
  • a humanized CD47 gene in some embodiments, comprises genetic material from a heterologous species (e.g., humans), wherein the humanized CD47 gene encodes a CD47 protein that comprises the encoded portion of the genetic material from the heterologous species.
  • a humanized CD47 gene of the present invention comprises genomic DNA of a heterologous species that encodes the extracellular portion of a CD47 protein that is expressed on the plasma membrane of a cell.
  • a humanized CD47 gene of the present invention comprises genomic DNA of a heterologous species that encodes the extracellular portion and the transmembrane portion of a CD47 protein that is expressed on the plasma membrane of a cell.
  • Non-human animals, embryos, cells and targeting constructs for making non-human animals, non- human embryos, and cells containing said humanized CD47 gene are also provided.
  • an endogenous CD47 gene is deleted.
  • an endogenous CD47 gene is altered, wherein a portion of the endogenous CD47 gene is replaced with a heterologous sequence (e.g., a human CD47 sequence, in whole or in part). In some embodiments, all or substantially all of an endogenous CD47 gene is replaced with a heterologous gene (e.g., a human CD47 gene). In some embodiments, a heterologous sequence (e.g., a human CD47 gene).
  • a portion of a heterologous CD47 gene is inserted into an endogenous non- human CD47 gene at an endogenous CD47 locus.
  • the heterologous gene is a human gene.
  • the modification or humanization is made to one of the two copies of the endogenous CD47 gene, giving rise to a non-human animal that is heterozygous with respect to the humanized CD47 gene.
  • a non-human animal is provided that is homozygous for a humanized CD47 gene.
  • a non-human animal of the present invention contains a human CD47 gene, in whole or in part, at an endogenous non-human CD47 locus.
  • non-human animals can be described as having a heterologous CD47 gene.
  • the replaced, inserted, modified or altered CD47 gene at the endogenous CD47 locus can be detected using a variety of methods including, for example, PCR, Western blot, Southern blot, restriction fragment length polymorphism (RFLP), or a gain or loss of allele assay.
  • the non-human animal is heterozygous with respect to the humanized CD47 gene.
  • the non-human animal is homozygous for the humanized CD47 gene.
  • a humanized CD47 gene according to the present invention includes a CD47 gene that has a second, third, fourth, fifth, sixth and seventh exon each having a sequence at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to a second, third, fourth, fifth, sixth and seventh exon that appear in a human CD47 gene of Table 3.
  • a second, third, fourth, fifth, sixth and seventh exon each having a sequence at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to a second, third, fourth, fifth, sixth and seventh exon that appear in a human CD47 gene of Table 3.
  • a humanized CD47 gene according to the present invention includes a CD47 gene that has a first exon and exon(s) downstream of exon 7 (e.g., eighth and ninth exons of isoforai 2) each having a sequence at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to a respective exon that appears in a mouse CD47 gene of Table 3.
  • exon 7 e.g., eighth and ninth exons of isoforai 2
  • a humanized CD47 gene according to the present invention includes a CD47 gene that has a 5' untranslated region and a 3' untranslated region each having a sequence at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to a 5' untranslated region and a 3' untranslated region that appear in a mouse CD47 gene of Table 3.
  • 50% e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
  • a humanized CD47 gene according to the present invention includes a CD47 gene that has a nucleotide coding sequence (e.g., a cDNA sequence) at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to a nucleotide coding sequence that appears in a human CD47 nucleotide coding sequence of Table 3.
  • a nucleotide coding sequence e.g., a cDNA sequence
  • a humanized CD47 protein produced by a non-human animal of the present invention has an extracellular portion having an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to an extracellular portion of a human CD47 protein that appears in Table 3.
  • a humanized CD47 protein produced by a non-human animal of the present invention has an extracellular portion having an amino acid sequence that is identical to amino acid residues 19-141 that appear in a human CD47 protein of Table 3.
  • a humanized CD47 protein produced by a non-human animal of the present invention has an N-terminal immunoglobulin V domain having an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to an N- terminal immunoglobulin V domain of a human CD47 protein that appears in Table 3.
  • a humanized CD47 protein produced by a non-human animal of the present invention has an N-terminal immunoglobulin V domain having an amino acid sequence that is identical to amino acid residues 19-127 that appear in a human CD47 protein of Table 3.
  • a humanized CD47 protein produced by a non-human animal of the present invention has an N-terminal immunoglobulin V domain and five transmembrane domains each having a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to an N-terminal immunoglobulin V domain and five transmembrane domains of a human CD47 protein that appears in Table 3.
  • 50% e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
  • a humanized CD47 protein produced by a non-human animal of the present invention has an intracytoplasmic tail having a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to an intracytoplasmic tail of a mouse CD47 protein that appears in Table 3.
  • a humanized CD47 protein produced by a non-human animal of the present invention has an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to amino acid residues 16-292 that appear in a human CD47 protein of Table 3.
  • a humanized CD47 protein produced by a non-human animal of the present invention has an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to amino acid residues 19-292 that appear in a human CD47 protein of Table 3.
  • a humanized CD47 protein produced by a non-human animal of the present invention has an amino acid sequence that is identical to amino acid residues 19-292 (or 16-292) that appear in a human CD47 protein of Table 3.
  • a humanized CD47 protein produced by a non-human animal of the present invention has an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to an amino acid sequence of a humanized CD47 protein that appears in Table 3.
  • a humanized CD47 protein produced by a non-human animal of the present invention has an amino acid sequence that is identical to an amino acid sequence of a humanized CD47 protein that appears in Table 3.
  • compositions and methods for making non-human animals that express a humanized CD47 protein including specific polymorphic forms, allelic variants (e.g., single amino acid differences) or alternatively spliced isoforms, are provided, including compositions and methods for making non-human animals that express such proteins from a human promoter and a human regulatory sequence.
  • the methods include inserting the genetic material encoding a human CD47 protein in whole or in part at a precise location in the genome of a non-human animal that corresponds to an endogenous CD47 gene thereby creating a humanized CD47 gene that expresses a CD47 protein that is human in whole or in part.
  • the methods include inserting genomic DNA corresponding to exons 2-7 of a human CD47 gene into an endogenous CD47 gene of the non-human animal thereby creating a humanized gene that encodes a CD47 protein that contains a human portion containing amino acids encoded by the inserted exons.
  • the coding region of the genetic material or polynucleotide sequence(s) encoding a human CD47 protein in whole or in part may be modified to include codons that are optimized for expression in the non-human animal (e.g., see U.S. Patent No.'s 5,670,356 and 5,874,304).
  • Codon optimized sequences are synthetic sequences, and preferably encode the identical polypeptide (or a biologically active fragment of a full length polypeptide which has substantially the same activity as the full length polypeptide) encoded by the non-codon optimized parent polynucleotide.
  • the coding region of the genetic material encoding a human CD47 protein may include an altered sequence to optimize codon usage for a particular cell type (e.g., a rodent cell).
  • a particular cell type e.g., a rodent cell
  • the codons of the genomic DNA corresponding to exons 2-7 of a human CD47 gene to be inserted into an endogenous CD47 gene of a non-human animal may be optimized for expression in a cell of the non-human animal.
  • a sequence may be described as a codon-optimized sequence.
  • a humanized CD47 gene approach employs a relatively minimal modification of the endogenous gene and results in natural CD47-mediated signal transduction in the non- human animal, in various embodiments, because the genomic sequence of the CD47 sequences are modified in a single fragment and therefore retain normal functionality by including necessary regulatory sequences.
  • the CD47 gene modification does not affect other surrounding genes or other endogenous CD47- interacting genes (e.g., thrombospondin, SIRPs, integrins, etc.).
  • the modification does not affect the assembly of a functional CD47 transmembrane protein on the plasma membrane and maintains normal effector functions via binding and subsequent signal transduction through the cytoplasmic portion of the protein which is unaffected by the modification.
  • FIG. 1 An exemplary method for humanizing an endogenous murine CD47 gene using a genomic fragment containing exons 2-7 of a human CD47 gene is provided in Figure 2.
  • genomic DNA containing exons 2-7 of a human CD47 gene is inserted into an endogenous murine CD47 gene locus by a targeting construct.
  • This genomic DNA includes the portion of the gene that encodes an extracellular portion and transmembrane domains (e.g., amino acid resides 16-292) of a human CD47 protein responsible for ligand binding.
  • a non-human animal having a humanized CD47 gene at the endogenous CD47 locus can be made by any method known in the art.
  • a targeting vector can be made that introduces a human CD47 gene in whole or in part with a selectable marker gene.
  • Figure 2 illustrates an endogenous CD47 locus of a mouse genome comprising an insertion of exons 2-7 of a human CD47 gene.
  • the targeting construct contains a 5' homology arm containing sequence upstream of exon 2 of an endogenous murine CD47 gene (-39 Kb), followed by a genomic DNA
  • the targeting construct contains a self-deleting drug selection cassette (e.g., a neomycin resistance gene flanked by loxP sequences; see U.S. Patent No.'s 8,697,851, 8,518,392 and 8,354,389, all of which are incorporated herein by reference).
  • exons 2-7 of an endogenous murine CD47 gene are replaced by the sequence contained in the targeting vector (i.e., exons 2-7 of a human CD47 gene).
  • a humanized CD47 gene is created resulting in a cell or non-human animal that expresses a humanized CD47 protein that contains amino acids encoded by exons 2-7 of a human CD47 gene.
  • the drug selection cassette is removed in a development- dependent manner, i.e., progeny derived from mice whose germ line cells containing the humanized CD47 gene described above will shed the selectable marker from differentiated cells during development.
  • non-human animals of the present invention may be prepared as described above, or using methods known in the art, to comprise additional human or humanized genes, oftentimes depending on the intended use of the non-human animal. Genetic material of such additional human or humanized genes may be introduced through the further alteration of the genome of cells (e.g., embryonic stem cells) having the genetic modifications as described above or through breeding techniques known in the art with other genetically modified strains as desired.
  • non-human animals of the present invention are prepared to further comprise one or more human or humanized genes selected from SIRPa (CD 172a), IL-3, M-CSF, GM-CSF and TPO.
  • non-human animals of the present invention may be prepared by introducing a targeting vector, as described herein, into a cell from a modified strain.
  • a targeting vector as described above, may be introduced into a mouse that is Rag2-deficient and IL-2Ry-deficient and include four human cytokines (Rag2 ⁇ / ⁇ IL2Ryc ⁇ / ⁇ ; M-CSF Hu ; IL-3/GM-CSF Hu ; hSIRPa tg ; TPO Hu ).
  • non-human animals of the present invention are prepared to further comprise a human or humanized signal-regulatory protein alpha (SIRPa) gene.
  • SIRPa signal-regulatory protein alpha
  • non-human animals of the present invention comprise a humanized CD47 gene, as described herein, and genetic material from a heterologous species (e.g., humans), wherein the genetic material encodes, in whole or in part, one or more heterologous proteins selected from SIRPa (CD172a), IL-3, M-CSF, GM-CSF and TPO.
  • non-human animals of the present invention comprise a humanized CD47 gene as described herein and genetic material from a heterologous species (e.g., humans), wherein the genetic material encodes, in whole or in part, a heterologous (e.g., human) SIRPa (CD 172a) protein.
  • non-human animals of the present invention further comprise a SIRPa gene that comprises an endogenous portion and a human portion (e.g., exons 2-4 of a human SIRPa gene), wherein the human portion encodes the extracellular domain of a SIRPa protein (e.g., amino acids corresponding to residues 28-362 of a human SIRPa protein) and the endogenous portion encodes the intracellular domain of an endogenous SIRPa protein; in some embodiments, the human portion and the endogenous portion are operably linked to an endogenous SIRPa promoter.
  • a SIRPa gene that comprises an endogenous portion and a human portion (e.g., exons 2-4 of a human SIRPa gene), wherein the human portion encodes the extracellular domain of a SIRPa protein (e.g., amino acids corresponding to residues 28-362 of a human SIRPa protein) and the endogenous portion encodes the intracellular domain of an endogenous SIRPa protein; in some embodiments, the human
  • non-human animals comprising a humanized CD47 gene may further comprise (e.g., via cross-breeding or multiple gene targeting strategies) one or more modifications as described inPCT/US2010/051339, filed October 4, 2010; PCT/US2013/058448, filed September 6, 2013; PCT/US2013/045788, filed June 14, 2013; PCT/US2014/056910, filed September 23, 2014; PCT/US2014/060568, filed October 15, 2014; the PCT/US2012/025040, filed February 14, 2014;
  • a rodent comprising a humanized CD47 gene (i.e., exons 2-7 of a human CD47 gene operably linked to exon 1 and exon 8 (and hence any downstream exons) of an endogenous rodent CD47 gene so that the humanized CD47 gene encodes a CD47 polypeptide having an extracellular portion from a human CD47 protein and an intracellular portion from a rodent CD47 protein) is crossed to a rodent comprising a humanized SIRPa gene (e.g., exons 2-4 of a human SIRPa gene operably linked to exons 1 and 5-8 of an endogenous rodent SIRPa gene so that the humanized SIRPa gene encodes a SIRPa polypeptide having an extracellular portion from a human SIRP
  • a humanized CD47 gene in a mouse i.e., a mouse with a CD47 gene that encodes a CD47 protein that includes a human portion and a mouse portion
  • other non-human animals that comprise a humanized CD47 gene are also provided.
  • such non-human animals comprise a humanized CD47 gene operably linked to an endogenous CD47 promoter.
  • such non-human animals express a humanized CD47 protein from an endogenous locus, wherein the humanized CD47 protein comprises amino acid residues 16-292 (or 19-141 or 19-127) of a human CD47 protein.
  • non-human animals include any of those which can be genetically modified to express a CD47 protein as disclosed herein, including, e.g., mammals, e.g., mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey), etc.
  • mammals e.g., mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey), etc.
  • suitable genetically modifiable ES cells are not readily available
  • other methods are employed to make a non- human animal comprising the genetic modification.
  • Such methods include, e.g., modifying a non-ES cell genome (e.g., a fibroblast or an induced pluripotent cell) and employing somatic cell nuclear transfer (SCNT) to transfer the genetically modified genome to a suitable cell, e.g., an enucleated oocyte, and gestating the modified cell (e.g., the modified oocyte) in a non-human animal under suitable conditions to form an embryo.
  • a non-ES cell genome e.g., a fibroblast or an induced pluripotent cell
  • SCNT somatic cell nuclear transfer
  • Methods for modifying a non-human animal genome include, e.g., employing a zinc finger nuclease (ZFN) or a transcription activator-like effector nuclease (TALEN) to modify a genome to include a humanized CD47 gene.
  • ZFN zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • a non-human animal of the present invention is a mammal.
  • a non-human animal of the present invention is a small mammal, e.g., of the superfamily Dipodoidea or Muroidea.
  • a genetically modified animal of the present invention is a rodent.
  • a rodent of the present invention is selected from a mouse, a rat, and a hamster.
  • a rodent of the present invention is selected from the superfamily Muroidea.
  • a genetically modified animal of the present invention is from a family selected from Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamster, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae (climbing mice, rock mice, with-tailed rats, Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., mole rates, bamboo rats, and zokors).
  • Calomyscidae e.g., mouse-like hamsters
  • Cricetidae e.g., hamster, New World rats and mice, voles
  • Muridae true mice and rats, gerbils
  • a genetically modified rodent of the present invention is selected from a true mouse or rat (family Muridae), a gerbil, a spiny mouse, and a crested rat. In some certain embodiments, a genetically modified mouse of the present invention is from a member of the family Muridae. In some embodiment, a non- human animal of the present invention is a rodent. In some certain embodiments, a rodent of the present invention is selected from a mouse and a rat. In some embodiments, a non- human animal of the present invention is a mouse.
  • a non-human animal of the present invention is a rodent that is a mouse of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa,
  • a mouse of the present invention is a 129 strain selected from the group consisting of a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129Sl/SvIm), 129S2, 129S4, 129S5, 129S9/SvEvH, 129/SvJae, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et al., 1999, Mammalian Genome 10:836; Auerbach, W.
  • a genetically modified mouse of the present invention is a mix of an aforementioned 129 strain and an aforementioned C57BL/6 strain. In some certain embodiments, a mouse of the present invention is a mix of aforementioned 129 strains, or a mix of aforementioned BL/6 strains. In some certain embodiments, a 129 strain of the mix as described herein is a 129S6 (129/SvEvTac) strain. In some embodiments, a mouse of the present invention is a BALB strain, e.g., BALB/c strain. In some embodiments, a mouse of the present invention is a mix of a BALB strain and another aforementioned strain.
  • a non-human animal of the present invention is a rat.
  • a rat of the present invention is selected from a Wistar rat, an LEA strain, a Sprague Dawley strain, a Fischer strain, F344, F6, and Dark Agouti.
  • a rat strain as described herein is a mix of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.
  • CD47 mutant and transgenic non-human animals e.g., miniature swine
  • cells have been reported (Koshimizu H. et al., (2014) PLoS One, 9(2):e89584; Lavender, K.J. et al. (2014) J. Immunol. Methods, 407:127-134; Tena, A. et al., (2014) Am. J.
  • Such animals have been employed in a variety of assays to determine, for example, the molecular aspects of CD47 expression, function and regulation. Considerable species differences have been discovered. Indeed, nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice express a SIRPa protein that is capable of interacting with human CD47 and, therefore, have been used extensively for the development of mouse models with components of the human immune system (e.g., see Takenaka, K. et al., (2007) Nat.
  • mice are not representative of the SIRPa allele present in other mouse strains and, generally, there is little cross- reaction between CD47 and SIRPa between species.
  • CD47 on mouse cells has been reported to have a near-complete mobility, while CD47 on human cells demonstrate only about 30-40% (Bruce, L. et al., (2003) Blood 101 :4180-4188; Mouro-Chanteloup, L. et al., (2000) VoxSanguinis 78:P030; Mouro-Chanteloup, L. et al., (2003) Blood 101 :338-344).
  • NOD/SCID mice are not without limitation.
  • mice represent an inefficient in vivo system for elucidation of CD47-dependent functions in various biological processes such as, for example, engraftment and phagocytosis. Further, existing mice represent a suboptimal in vivo system for development of CD47 targeted therapies.
  • Non-human animals of the present invention provide an improved in vivo system and source of biological materials (e.g., cells) expressing human CD47 that are useful for a variety of assays.
  • non-human animals of the present invention are used to develop therapeutics that target CD47 and/or modulate CD47-SIRPa signaling.
  • non-human animals of the present invention are used to screen and develop candidate therapeutics (e.g., antibodies) that bind human CD47.
  • candidate therapeutics e.g., antibodies
  • non-human animals of the present invention are used to determine the binding profile of antagonists and/or agonists of a humanized CD47 on the surface of a cell of a non-human animal as described herein. In some embodiments, non- human animals of the present invention are used to determine the epitope or epitopes of one or more candidate therapeutic antibodies that bind human CD47.
  • non-human animals of the present invention are used to determine the pharmacokinetic profiles of anti-CD47 antibodies.
  • one or more non-human animals of the present invention and one or more control or reference non-human animals are each exposed to one or more candidate therapeutic anti- CD47 antibody at various doses (e.g., 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/mg, 7.5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg or more).
  • various doses e.g., 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/mg, 7.5 mg/kg, 10 mg/kg, 15 mg/
  • Candidate therapeutic antibodies may be dosed via any desired route of administration (e.g., subcutaneously, intravenously, intramuscular, intraperitoneal, etc.). Blood is isolated from non-human animals (humanized and control) at various time points (e.g., 0 hr, 6 hr, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or up to 30 or more days). Various assays may be performed to determine the pharmacokinetic profiles of administered candidate therapeutic antibodies using samples obtained from non-human animals as described herein including, but not limited to, total IgG, anti-therapeutic antibody response, agglutination, etc.
  • non-human animals of the present invention are used to measure the therapeutic effect of blocking or modulating CD47 signaling and the effect on gene expression as a result of cellular changes.
  • a non-human animal of the present invention or cells isolated therefrom are exposed to a candidate therapeutic that binds to a humanized CD47 protein (or a human portion of a CD47 protein) on the surface of a cell of the non-human animal and, after a subsequent period of time, analyzed for effects on CD47-dependent processes, for example, adhesion, angiogenesis, apoptosis, inflammation, migration, phagocytosis, proliferation and clearance of tumors (or tumor cells).
  • Non-human animals of the present invention express humanized CD47 protein, thus cells, cell lines, and cell cultures can be generated to serve as a source of humanized CD47 for use in binding and functional assays, e.g., to assay for binding or function of a CD47 antagonist or agonist, particularly where the antagonist or agonist is specific for a human SIRPa sequence or epitope.
  • CD47 epitopes bound by candidate therapeutic antibodies can be determined using cells isolated from non-human animals of the present invention.
  • Cells from non-human animals of the present invention can be isolated and used on an ad hoc basis, or can be maintained in culture for many generations.
  • cells from a non-human animal of the present invention are immortalized and maintained in culture indefinitely (e.g., in serial cultures).
  • cells and/or non-human animals of the present invention are used in a survival and/or proliferation assay (e.g., employing B or T cells) to screen and develop candidate therapeutics that modulate human CD47 signaling.
  • Activation or loss of CD47 can play an important role in the regulation of cell proliferation, and induction of apoptosis by CD47 may result from the activation of specific epitopes of the extracellular domain of CD47, therefore, candidate CD47 modulators (e.g., antagonists or agonists) may be identified, characterized and developed using cells of non-human animals of the present invention and/or a non-human animal as described herein.
  • cells and/or non-human animals of the present invention are used in survivalor death assay(s) to determine the effect on proliferation or apoptosis of a specific cell(s) (e.g., cancer cells) in the presence and absence of CD47.
  • a specific cell(s) e.g., cancer cells
  • cells and/or non-human animals of the present invention are used in xenotransplantation of heterologous (e.g., human) cells to determine the CD47- mediated functions in the physiological (e.g., immune) response to the transplanted human cells.
  • candidate therapeutics that bind, or block one or more functions of, human CD47 are characterized in a non-human animal of the present invention. Suitable measurements include various cellular assays, proliferation assays, serum immunoglobulin analysis (e.g., antibody titer), cytotoxicity assays, and
  • non-human animals of the present invention are used to characterize the CD47-mediated functions regulating an immune response to an antigen.
  • the antigen is associated with a neoplasm.
  • the antigen is associated with an autoimmune disease or condition.
  • the antigen is a target associated with a disease or condition suffered by one or more human patients in need of treatment.
  • non-human animals of the present invention are used in transplantation or adoptive transfer experiments to determine the therapeutic potential of compounds or biological agents to modulate CD47-dependent regulation of new lymphocytes and their immune function.
  • non-human animals of the present invention are transplanted with human B cells.
  • cells of non-human animals of the present invention are used in a cell migration or spreading assay to screen and develop candidate therapeutics that modulate human CD47.
  • Such processes are necessary for many cellular processes including wound healing, differentiation, proliferation and survival.
  • cells of non-human animals of the present invention are used in phagocytosis assays to determine the therapeutic potential of compounds or biological agents to modulate CD47-dependent regulation of phagocytosis.
  • cells of non-human animals of the present invention are used in tumor cell growth (or proliferation) assays to determine the therapeutic potential of compounds or biological agents to modulate CD47-dependent regulation and/or apoptosis of tumor cells.
  • an inflammatory disease or condition is induced in one or more non-human animals of the present invention to provide an in vivo system for determining the therapeutic potential of compounds or biological agents to modulate CD47-dependent regulation of one or more functions of the inflammatory disease or condition.
  • the inflammatory disease or condition is associated with a neoplasm.
  • an anti-angiogenic condition is induced in one or more non-human animals of the present invention to provide an in vivo system for determining the therapeutic potential of compounds or biological agents to modulate CD47-dependent regulation of one or more functions of the anti-angiogenic condition.
  • exemplary functions that can be evaluated to determine therapeutic efficacy include chemokine expression, nitric oxide (NO)- stimulated responses and blood flow recovery.
  • Non-human animals of the present invention provide an in vivo system for the analysis and testing of a drug or vaccine.
  • a candidate drug or vaccine may be delivered to one or more non-human animals of the present invention, followed by monitoring of the non-human animals to determine one or more of the immune response to the drug or vaccine, the safety profile of the drug or vaccine, or the effect on a disease or condition.
  • Exemplary methods used to determine the safety profile include measurements of toxicity, optimal dose concentration, efficacy of the drug or vaccine, and possible risk factors.
  • Such drugs or vaccines may be improved and/or developed in such non-human animals.
  • Non-human animals of the present invention provide an in vivo system for assessing the pharmacokinetic properties of a drug targeting CD47.
  • a drug targeting CD47 may be delivered or administered to one or more non-human animals of the present invention, followed by monitoring of, or performing one or more assays on, the non-human animals (or cells isolated therefrom) to determine the effect of the drug on the non-human animal.
  • Pharmacokinetic properties include, but are not limited to, how an animal processes the drug into various metabolites (or detection of the presence or absence of one or more drug metabolites, including, toxic metabolites), drug half-life, circulating levels of drug after administration (e.g., serum concentration of drug), anti-drug response (e.g., anti-drug antibodies), drug absorption and distribution, route of administration, routes of excretion and/or clearance of the drug.
  • pharmacokinetic and pharmacodynamic properties of drugs are monitored in or through the use of non-human animals of the present invention.
  • Non-human animals of the present invention provide an in vivo system for assessing the on- target toxicity of a drug targeting CD47.
  • a drug targeting CD47 may be delivered or administered to one or more non-human animals of the present invention, followed by monitoring of or performing one or more assays on the non-human animals (or cells isolated therefrom) to determine the on-target toxic effect of the drug on the non-human animal.
  • drugs are intended to modulate one or more functions of their targets.
  • a CD47 modulator is intended to modulate CD47-mediated functions (e.g., CD47 induced apoptosis) through interacting in some way with the CD47 molecule on the surface of one or more cells.
  • such a modulator may have an adverse effect that is an exaggeration of the desired pharmacologic action(s) of the modulator.
  • Such effects are termed on-target effects.
  • Exemplary on-target effects includetoo high of a dose, chronic
  • on- target effects of a drug targeting CD47 identified in or through the use of non-human animals of the present invention are used to determine a previously unknown function(s) of CD47.
  • Non-human animals of the present invention provide an in vivo system for assessing the off-target toxicity of a drug targeting CD47.
  • a drug targeting CD47 may be delivered or administered to one or more non-human animals of the present invention, followed by monitoring of or performing one or more assays on the non-human animals (or cells isolated therefrom) to determine the off-target toxic effect of the drug on the non-human animal.
  • Off-target effects can occur when a drug interacts with an unintended target (e.g., cross-reactivity to a common epitope). Such interactions can occur in an intended or unintended tissue.
  • mirror image isomers (enantiomers) of a drug can lead to off-target toxic effects.
  • a drug can be delivered or administered to one or more non-human animals of the present invention, followed by monitoring of or performing one or more assays on the non-human animals (or cells isolated therefrom) to determine the off-target toxic effect of the drug on the non-human animal.
  • Off-target effects can occur when
  • off-target effects include incorrect activation/inhibition of an incorrect target regardless of the tissue in which the incorrect target is found.
  • off- target effects of a drug targeting CD47 are determined by comparing the effects of administering the drug to non-human animals of the present invention to one or more reference non-human animals.
  • performing an assay includes determining the effect on the phenotype (e.g., change in body weight) and/or genotype of the non-human animal to which the drug is administered. In some embodiments, performing an assay includes determining lot-to-lot variability for a CD47 modulator (e.g., an antagonist or an agonist). In some embodiments, performing an assay includes determining the differences between the effects of a drug targeting CD47 administered to a non-human animal of the present invention and a reference non-human animal.
  • a CD47 modulator e.g., an antagonist or an agonist
  • reference non- human animals may have a modification as described herein, a modification that is different as described herein (e.g., one that has a disruption, deletion or otherwise nonfunctional CD47 gene) or no modification (i.e., a wild-type non-human animal).
  • Exemplary parameters that may be measured in non-human animals (or in and/or using cells isolated therefrom) for assessing the pharmacokinetic properties, on-target toxicity, and/or off-target toxicity of a drug targeting CD47 include, but are not limited to, agglutination, autophagy, cell division, cell death, complement-mediated hemolysis, DNA integrity, drug-specific antibody titer, drug metabolism, gene expression arrays, hematocrit levels, hematuria, metabolic activity, mitochondrial activity, oxidative stress, phagocytosis, protein biosynthesis, protein degradation, protein secretion, stress response, target tissue drug concentration, non-target tissue drug concentration, transcriptional activity and the like.
  • non-human animals of the present invention are used to determine a pharmaceutically effective dose of a CD47 modulator.
  • Non-human animals of the present invention provide an improved in vivo system for the development and characterization of candidate therapeutics for use in cancer.
  • non-human animals of the present invention may be implanted with a tumor, followed by administration of one or more candidate therapeutics.
  • candidate therapeutics may include a multi- specific antibody (e.g., a bi- specific antibody) or an antibody cocktail; in some embodiments, candidate therapeutics include combination therapy such as, for example, administration of mono-specific antibodies dosed sequentially or simultaneously.
  • the tumor may be allowed sufficient time to be established in one or more locations within the non-human animal. Tumor cell proliferation, growth, etc. may be measured both before and after administration with the candidate therapeutic(s). Cytotoxicity of candidate therapeutics may also be measured in the non-human animal as desired.
  • Non-human animals of the present invention provide improved in vivo system elucidating mechanisms of human cell-to-cell interaction through adoptive transfer.
  • non-human animals of the present invention may by implanted with a tumor xenograft, followed by a second implantation of tumor infiltrating lymphocytes could be implanted in the non-human animals by adoptive transfer to determine the effectiveness in eradication of solid tumors or other malignancies.
  • Such experiments may be done with human cells due to the exclusive presence of human CD47 without competition with endogenous CD47 of the non-human animal.
  • such experiments may include the use of mouse cells from a NOD/SCID or BRG (BALB/c Ragl ⁇ TL-lRyc "7" ) background.
  • therapies and pharmaceuticals for use in xenotransplantation can be improved and/or developed in such non-human animals.
  • Non-human animals of the present invention provide an improved in vivo system for maintenance and development of human hematopoietic stem cells through engraftment.
  • non-human animals of the present invention provide improved development and maintenance of human stem cells within the non-human animal.
  • increased populations of differentiated human B and T cells are observed in the blood, bone marrow, spleen and thymus of the non-human animal.
  • optimal T and NK cell homeostasis is observed in cells in the blood, bone marrow, spleen and thymus of the non-human animal.
  • non- human animals of the present invention demonstratean increase in the level or amount of red blood cells (RBCs) as compared to one or more reference non-human animals.
  • RBCs red blood cells
  • Non-human animals of the present invention can be employed to assess the efficacy of a therapeutic drug targeting human cells.
  • a non-human animal of the present invention is transplanted with human cells, and a drug candidate targeting such human cells is administered to such non-human animal.
  • the therapeutic efficacy of the drug is then determined by monitoring the human cells in the non-human animal after the administration of the drug.
  • Drugs that can be tested in the non-human animals include both small molecule compounds, i.e., compounds of molecular weights of less than 1500 kD, 1200 kD, 1000 kD, or 800 daltons, and large molecular compounds (such as proteins, e.g., antibodies), which have intended therapeutic effects for the treatment of human diseases and conditions by targeting (e.g., binding to and/or acting on) human cells.
  • small molecule compounds i.e., compounds of molecular weights of less than 1500 kD, 1200 kD, 1000 kD, or 800 daltons
  • large molecular compounds such as proteins, e.g., antibodies
  • the drug is an anti-cancer drug
  • the human cells are cancer cells, which can be cells of a primary cancer or cells of cell lines established from a primary cancer.
  • a non-human animal of the present invention is transplanted with human cancer cells, and an anti-cancer drug is given to the non-human animal.
  • the efficacy of the drug can be determined by assessing whether growth or metastasis of the human cancer cells in the non-human animal is inhibited as a result of the administration of the drug.
  • the anti-cancer drug is an antibody molecule, which binds to an antigen on human cancer cells.
  • the anti-cancer drug is a bi-specific antibody that binds to an antigen on human cancer cells, and to an antigen on other human cells, for example, cells of the human immune system (or "human immune cells") such as B cells and T cells.
  • a non-human animal of the present invention is engrafted with human immune cells or cells that differentiate into human immune cells.
  • Such non- human animal with engrafted human immune cells is transplanted with human cancer cells, and is administered an anti-cancer drug, such as a bi-specific antibody that binds to an antigen on human cancer cells and to an antigen on human immune cells (e.g., T cells).
  • an anti-cancer drug such as a bi-specific antibody that binds to an antigen on human cancer cells and to an antigen on human immune cells (e.g., T cells).
  • the therapeutic efficacy of the bi-specific antibody can be evaluated based on its ability to inhibit growth or metastasis of the human cancer cells in the non-human animal.
  • the non-human animal of the present invention is engrafted with human CD34 + hematopoietic progenitor cells which give rise to human immune cells (including T cells, B cells, NK cells, among others).
  • Human B cell lymphoma cells e.g., Raji cells
  • a bi-specific antibody that binds to tumor antigen (e.g., an antigen on normal B cells and certain B cell malignancies such as CD20) and to the CD3 subunit of the T cell receptor, to test the ability of the bi-specific antibody to inhibit tumor growth in the non-human animal.
  • tumor antigen e.g., an antigen on normal B cells and certain B cell malignancies such as CD20
  • This example illustrates exemplary methods of humanizing an endogenous gene encoding cluster of differentiation 47 (CD47) in a non-human mammal such as a rodent (e.g., a mouse).
  • a rodent e.g., a mouse
  • the methods described in this example can be employed to humanize an endogenous CD47 gene of a non-human animal using any human sequence, or
  • a human CD47 gene that appears in bacterial artificial chromosome (BAC) clone RP11- 69A17 is employed for humanizing an endogenous CD47 gene of a mouse.
  • extracellular N-terminal IgV domain and five transmembrane domains of an endogenous CD47 gene was constructed using VELOCIGENE® technology (see, e.g., U.S. Patent No. 6,586,251 and Valenzuela et al., (2003) High- throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21(6):652-659;
  • mouse bacterial artification chromosome (BAC) clone RP23-230L20 (Invitrogen) was modified to delete the sequence containing exons 2-7 of an endogenous CD47 gene and insert exons 2-7 of a human CD47 gene using human BAC clone RPl 1- 69A17 (Invitrogen), which encodes amino acids 16-292 of a human CD47 polypeptide.
  • Endogenous DNA containing genomic DNA corresponding to exons 1, 8 and 9 of isoform 2 as well as the 5' and 3' untranslated regions (UTRs) were retained.
  • genomic DNA corresponding to exons 2-7 of an endogenous CD47 gene (-30.8 kb) was replaced in BAC clone RP23-230L20 by homologous recombination in bacterial cells to insert a DNA fragment containing -23.9 kb of genomic human DNA corresponding to exons 2-7 of a human CD47 gene from BAC clone RP1 l-69A17and -4995 bp corresponding to a self- deleting neomycin cassette flanked by recombinase recognition sites (/oxP-hUbl-em7- Neo-pA-mPrml-Crei-/oxP; see U.S.
  • the targeting vector contained, from 5' to 3', a 5' homology arm containing -39 kb of mouse genomic DNA from BAC clone RP23-230L20, -29.3 kb of human genomic DNA from BAC clone RPl 1-69A17 (containing exons 2-7 of a human CD47 gene), a self-deleting neomycin cassette flanked by loxP sites, and -98.8 kb of mouse genomic DNA from BAC clone RP23-230L20.
  • a modifed RP23-230L20 BAC clone was created that resulted in a humanized CD47 gene which contained a mouse 5' UTR, a mouse exon 1, human exons 2-7, mouse exons 8-9 and a mouse 3'UTR.
  • Protein sequences of four projected alternatively spliced isoforms of humanized CD47 are provided in Table 3 which indicate the resulting mouse and human amino acids encoded by the mouse and human DNA, respectively.
  • ES cells mouse embryonic stem (ES) cells to create modified ES cells comprising an endogenous CD47 gene that is humanized from exons 2-7. Positively targeted ES cells containing a humanized CD47 gene were identified by an assay
  • nucleotide sequence across the downstream insertion point at the 5' end of the self- deleting neomycin cassette included the following, which indicates human CD47 genomic sequence contiguous with cassette sequence downstream of the insertion point (contained within the parenteses below with loxP sequence italicized):
  • the nucleotide sequence across the downstream insertion point at the 3' end of the neomycin cassette included the following, which indicates cassette sequence contiguous with mouse genomic sequence 3' of exon 7 of an endogenous CD47 gene (contained within the parentheses below with loxP sequence italicized):
  • nucleotide sequence across the downstream insertion point after deletion of the neomycin cassette included the following, which indicates human and mouse genomic sequence juxtaposed with remaining cassette sequence loxP sequence (contained within the parentheses below with loxP sequence italicized): GGATCCATTT TAAGTAATAG AATAGGATTT TTAATTGTTC
  • VELOCIMOUSE® method see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al., FO generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses, 2007, Nature Biotech. 25(l):91-99) to generate a litter of pups containing an insertion of exons 2-7 of a human CD47 gene into an endogenous CD47 gene of a mouse.
  • mice bearing the humanization of exons 2-7 of an endogenous CD47 gene were again confirmed and identified by genotyping of DNA isolated from tail snips using a modification of allele assay (Valenzuela et al., supra) that detected the presence of the human CD47 gene sequences. Pups are genotyped and cohorts of animals heterozygous for the humanized CD47 gene construct are selected for characterization.
  • Example 2 Expression of humanized CD47 polypeptide by mouse red blood cells.
  • Example 1 demonstrates that non-human animals (e.g., rodents) modified to contain a humanized CD47 gene according to Example 1 can be used to screen CD47 modulators (e.g., anti-CD47 antibodies) and determine various characterisitics such as, for example, pharamcokinetics and safety profiles.
  • CD47 modulators e.g., anti-CD47 antibodies
  • various characterisitics such as, for example, pharamcokinetics and safety profiles.
  • anti-CD47 antibodies are screened on mouse red blood cells (RBCs) isolated from rodents made in accordance with Example 1, which rodents express a humanized CD47 polypeptide as described herein.
  • the plate was incubated again on ice for 30 minutes, followed by washing once with staining buffer.
  • the cells in each well were resuspended in 200 ⁇ , of staining buffer and filtered through a 96-well filter plate.
  • the cells in the plate were analyzed using theBD
  • Example 3 Hemagglutination of mouse red blood cells expressing humanized CD47 polypeptide.
  • Example 1 further demonstrates that non-human animals (e.g., rodents) modified to contain a humanized CD47 gene according to Example 1 can be used in various assays (e.g., hemagglutination assay) to screen CD47 modulators (e.g., anti-CD47 antibodies) and determine various characterisitics such as, for example, pharamcokinetics and safety profiles.
  • CD47 modulators e.g., anti-CD47 antibodies
  • characterisitics such as, for example, pharamcokinetics and safety profiles.
  • several anti-CD47 antibodies are screened on mouse red blood cells (RBCs) that express a humanized CD47 polypeptide as described herein to determine antibody concentration that promotes hemagglutination.
  • RBCs from wild-type and humanized CD47 mice were prepared as described in Example 2. Twenty (20) ⁇ , of anti-CD47 antibody (at 5-fold serial dilution) was added into wells 1-12 across a 96-well V-bottom plate followed by the addition of 80 ⁇ , of 0.5% mouse RBCs to all wells of the plate. The plates were tapped gently to mix and incubated at room temperature (24-27°C) for 30 minutes. Agglutination endpoint was observed visually (i.e., RBCs settle to the bottom in negative samples, while RBCs agglutinate in possitive samples). Exemplary results are shown in Figure 5, with boxes to outline the wells that show heme-agglutination.
  • This Example illustrates a method of assessing the pharmacokinetic clearance of CD47 modulators (e.g., anti-CD47 antibodies) in non-human animals (e.g., rodents) modified to contain a humanized CD47 gene according to Example 1.
  • CD47 modulators e.g., anti-CD47 antibodies
  • non-human animals e.g., rodents
  • humanized CD47 rodents e.g., mice
  • serum levels of antibodies were determined using an ELISA assay.
  • mice homozygous for humanized CD47 were administered four anti-CD47 antibodies (Ab F, Ab G,Ab Hand Ab I) and an IgG4s isotype control antibody (IgG4s).
  • the genetic background of the mice were 75% CD57BL/6 and 25% 129Sv.
  • Each antibody was tested in five humanized CD47 rodents. All antibodies were administered subcutaneously at a dose of 50 mg/kg.
  • One pre- bleed was collected one day prior to administration of antibody (day 0).
  • Post-injection bleeds were collected at 6 hours, 1 day, 2 days, 3 days, 4 days, 7 days, 10 days and 14 days. Serum fractions from bleeds were separated and subjected to total human antibody analysis using an ELISA immunoassay.
  • concentrations in the sera were calculated based on the reference standard curve generated using Graphpad Prism software. Exemplary results are shown in Figure 6 and Table 6.
  • non-human animals of the present invention can be used to assess one or more pharmacokinetic properties of a drug targeting CD47 (e.g., an anti-CD47 antibody) such as, for example, circulating drug levels. Morever, non-human animals described herein can be used to assess the toxicity of a drug targeting CD47 by determining adverse effects after administration.
  • a drug targeting CD47 e.g., an anti-CD47 antibody
  • This Example illustrates a method of assessing the pharmacokinetic clearance of CD47 modulators (e.g., anti-CD47 antibodies) in non-human animals (e.g., rodents) modified to contain humanized CD47 (according to Example 1) and SIRPa genes.
  • CD47 modulators e.g., anti-CD47 antibodies
  • non-human animals e.g., rodents
  • SIRPa genes e.g., SIRPa genes
  • humanized CD47 rodents described herein were modifed to further contain a humanized SIRPa gene that contains an endogenous portion and a human portion, which human portion encodes the extracellular domain of a human SIRPa protein (e.g., amino acids 28-362 of a human SIRPa protein) and which endogenous portion encodes an intracellular domain of an endogenous SIRPa protein (e.g., amino acids encoding transmembrane and intracellular portions of a murine SIRPa protein) as described in PCT/US14/56910, filed September 23, 2014, which is incorporated herein by reference.
  • Double humanized CD47/SIRPa mice were made by breeding humanized SIRPa mice to humanized CD47 mice.
  • double humanized CD47/SIRPa rodents e.g., mice
  • various anti-CD47 antibodies e.g., mice
  • their corresponding pharmacokinetic profiles were determined.
  • the genetic background of the mice were 75% CD57BL/6 and 25% 129Sv. All antibodies were administered in a single subcutaneous dose of 50 mg/kg. One pre-bleed was collected one day prior to
  • Ab J had less of an effect on hematocrit levels than other anti-CD47 antibodies tested (Ab F, Ab G, Ab I, etc.) and comparable changes in hematocrit levels to control (hIgG4s) in CD47 hu/hu SIRPa hu/hu mice.
  • Measurements of hematocrit on days 2-4 showed the largest drop from normal range (-38.5-45.1%), which included groups administered Abs F, G and I.
  • a monovalent form of Ab F demonstrated a delayed lowering effect on hematocrit as compared to other antibodies tested.
  • the inventors reasoned that differences in hematocrit levels among the various treatment groups could be attributed to a difference in epitope recognized by the various antibodies.
  • mice dosed with selected anti-CD47 antibodies demonstrated positive urine dipstick tests for heme at 6 hours.
  • Ab J and Ab F treatment groups each had one mouse positive for heme on day 1, while all other timepoints were negative.No significant weight loss (>20%) was observed in any treatment group.
  • non-human animals of the present invention provide an in vivo system for assessing the pharmacokinetic properties and/or profiles of one or more drugs targeting CD47 (e.g., one or more anti-CD47 antibodies) such as, for example, circulating drug levels. Morever, non-human animals as described herein engineered to further contain other humanized genes (e.g., humanized SIRPa) can be used to assess the target-mediated clearance of one or more drugs targeting CD47.
  • drugs targeting CD47 e.g., one or more anti-CD47 antibodies
  • circulating drug levels e.g., circulating drug levels.
  • non-human animals as described herein engineered to further contain other humanized genes e.g., humanized SIRPa

Abstract

La présente invention concerne des animaux non humains et des procédés et des compositions permettant de les produire et de les utiliser, lesdits animaux non humains étant caractérisés par une humanisation d'un gène CD et, notamment, par une humanisation d'un gène CD47. Lesdits animaux non humains peuvent être décrits, dans certains modes de réalisation, comme présentant une modification génétique concernant un gène CD47 endogène, de sorte que lesdits animaux non humains expriment un polypeptide CD47 qui comprend une partie humaine et une partie non humaine (par exemple une partie murine).
PCT/US2015/062614 2014-12-05 2015-11-25 Animaux non humains possédant un gène cd47 humanisé WO2016089692A1 (fr)

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JP2017526681A JP6730277B2 (ja) 2014-12-05 2015-11-25 ヒト化されたｃｌｕｓｔｅｒ ｏｆ ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ ４７遺伝子を有する非ヒト動物
RS20190370A RS58536B1 (sr) 2014-12-05 2015-11-25 Ne-humane životinje koje imaju humanizovani gen klastera diferencijacije 47
IL286403A IL286403B2 (en) 2014-12-05 2015-11-25 Non-human animals possess a humanized CLUSTER OF DIFFERENTIATION 47 gene
DK15813960.0T DK3086637T3 (en) 2014-12-05 2015-11-25 NON-HUMAN ANIMALS WITH A HUMANIZED CLUSTER OF DIFFERENTIATION 47 GEN
RU2017123357A RU2728412C2 (ru) 2014-12-05 2015-11-25 Животные, отличные от человека, имеющие гуманизированный ген кластера дифференцировки 47
KR1020217032415A KR102508175B1 (ko) 2014-12-05 2015-11-25 인간화 분화 클러스터 47 유전자를 가진 비인간 동물
SG11201703463WA SG11201703463WA (en) 2014-12-05 2015-11-25 Non-human animals having a humanized cluster of differentiation 47 gene
AU2015355328A AU2015355328B2 (en) 2014-12-05 2015-11-25 Non-human animals having a humanized cluster of differentiation 47 gene
EP23192469.7A EP4296278A3 (fr) 2014-12-05 2015-11-25 Animaux non humains ayant un groupe humanisé de gène de différenciation 47
CN201580066047.0A CN107205368B (zh) 2014-12-05 2015-11-25 具有人源化分化簇47基因的非人动物
SI201530659T SI3086637T1 (sl) 2014-12-05 2015-11-25 Nehumane živali s humaniziranim označevalcem diferenciacije gena CD47
EP15813960.0A EP3086637B1 (fr) 2014-12-05 2015-11-25 Animaux non humains possédant un gène cd47 humanisé
MX2017007293A MX2017007293A (es) 2014-12-05 2015-11-25 Animales no humanos que tienen un gen del grupo de diferenciacion 47 humanizado.
BR112017010490A BR112017010490A2 (pt) 2014-12-05 2015-11-25 roedor, polipeptídeo cd47, célula ou tecido de roedor isolado, célula-tronco emprionária de roedor, embrião de roedor, e, métodos de produzir um roedor, de fornecimento de um roedor, de enxerto de células humanas em um roedor, de avaliação de eficácia terapêutica de uma droga direcionada a células humanas e de avaliar as propriedades farmacocinéticas, de avaliar a toxicidade específica e inespecífica de uma droga direcionada ao cd47 humano.
EP18207075.5A EP3466255B1 (fr) 2014-12-05 2015-11-25 Animaux non humains ayant un groupe humanisé de gène de différenciation 47
EP20215046.2A EP3850946B1 (fr) 2014-12-05 2015-11-25 Animaux non humains ayant un groupe humanisé de gène de différenciation 47
PL18207075T PL3466255T3 (pl) 2014-12-05 2015-11-25 Zwierzęta inne niż człowiek mające humanizowany gen kompleksu różnicowania 47
KR1020177013948A KR102313073B1 (ko) 2014-12-05 2015-11-25 인간화 분화 클러스터 47 유전자를 가진 비인간 동물
LTEP15813960.0T LT3086637T (lt) 2014-12-05 2015-11-25 Gyvūnai, išskyrus žmogų, turintys humanizuota diferenciacijos klasterio 47 geną
PL15813960T PL3086637T3 (pl) 2014-12-05 2015-11-25 Zwierzęta nie będące człowiekiem mające humanizowany gen kompleksu różnicowania 47
NZ731471A NZ731471A (en) 2014-12-05 2015-11-25 Non-human animals having a humanized cluster of differentiation 47 gene
KR1020237007814A KR102617691B1 (ko) 2014-12-05 2015-11-25 인간화 분화 클러스터 47 유전자를 가진 비인간 동물
CA2967834A CA2967834C (fr) 2014-12-05 2015-11-25 Animaux non humains possedant un gene cd47 humanise
ES15813960T ES2716735T3 (es) 2014-12-05 2015-11-25 Animales no humanos que tienen un gen del grupo de diferenciación 47 humanizado
IL252019A IL252019B (en) 2014-12-05 2017-04-30 Non-human animals possessing a cluster of differentiation 47 gene of human origin
CY20191100334T CY1121547T1 (el) 2014-12-05 2019-03-21 Μη ανθρωπινα ζωα που εχουν ενα εξανθρωποποιημενο γονιδιο συμπλεγματος διαφοροποιησης 47
HRP20190634TT HRP20190634T1 (hr) 2014-12-05 2019-04-02 Životinje koje nisu ljudi, koje imaju humanizirani gen 47 iz razlikovnog grozda
CY20211100382T CY1124089T1 (el) 2014-12-05 2021-05-06 Μη ανθρωπινα ζωα που εχουν ενα εξανθρωπισμενο γονιδιο συμπλεγματος διαφοροποιησης 47
AU2022203787A AU2022203787A1 (en) 2014-12-05 2022-06-02 Non-human animals having a humanized cluster of differentiation 47 gene

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